Early-life exposure to estrogenic compounds affects the development of the reproductive system in rodent models and humans. Soy products, which contain phytoestrogens such as genistein, are one source of exposure in infants fed soy formula, and they result in high serum concentrations.Our goal was to determine whether soy exposure is associated with differential DNA methylation in vaginal cells from soy-fed infant girls.Using the Illumina HumanMethylation450 BeadChip, we evaluated epigenome-wide DNA methylation in vaginal cells from four soy formula-fed and six cow formula-fed girls from the Infant Feeding and Early Development (IFED) study. Using pyrosequencing we followed up the two most differentially methylated sites in 214 vaginal cell samples serially collected between birth and 9 months of age from 50 girls (28 soy formula-fed and 22 cow formula-fed). With a mouse model, we examined the effect of neonatal exposure to genistein on gene specific mRNA levels in vaginal tissue.The epigenome-wide scan suggested differences in methylation between soy formula-fed and cow formula-fed infants at three CpGs in the gene proline rich 5 like (PRR5L) (p < 104). Pyrosequencing of the two feeding groups found that methylation levels progressively diverged with age, with pointwise differences becoming statistically significant after 126 days. Genistein-exposed mice showed a 50% decrease in vaginal Prr5l mRNA levels compared to controls.Girls fed soy formula have altered DNA methylation in vaginal cell DNA which may be associated with decreased expression of an estrogen-responsive gene. Citation: Harlid S, Adgent M, Jefferson WN, Panduri V, Umbach DM, Xu Z, Stallings VA, Williams CJ, Rogan WJ, Taylor JA. 2017. Soy formula and epigenetic modifications: analysis of vaginal epithelial cells from infant girls in the IFED study. Environ Health Perspect 125:447-452; http://dx.doi.org/10.1289/EHP428.